Methods of expanding hematopoietic stem cells, compositions, and methods of use thereof

ABSTRACT

Described herein are methods of expanding a population of hematopoietic stem cells by contacting the population of hematopoietic stem cells with an effective amount of an inhibitor of G-protein coupled receptor 65 (GPR65) and providing a population of expanded, substantially undifferentiated hematopoietic stem cells. Exemplary GPR65 inhibitors include siRNAs and certain sphingolipids. Also described are populations of expanded hematopoietic stem cells produced by the methods, media and kits containing GPR65 inhibitors, and methods for administering an expanded population of hematopoietic stem cells to patients.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/US2016/053278,filed Sep. 23, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/222,921, filed Sep. 24, 2015, both of which areincorporated by reference in their entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under DK068634 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE DISCLOSURE

The present disclosure is related to methods of stimulating and/orexpanding hematopoietic stem cells by inhibiting suppressors ofhematopoietic stem cells.

BACKGROUND

Bone marrow stem cells have been used to treat a variety of diseasesincluding leukemia, multiple myeloma, some types of lymphoma, graftversus host disease, and genetic disorders of the blood and immunesystem including aplastic anemia, sickle cell anemia, Severe CombinedImmune Deficiency (SCID), Wiskott-Aldrich Syndrome (WAS), IPEX Syndrome,Hemophagocytic Lymphohistiocytosis (HLH), X-linked LymphoproliferativeDisease (XLP) and Chronic Granulomatous Disease (CGD). However, it isdifficult and time-consuming to find a matching donor for a particularpatient. Only a fraction of patients will find a suitable donor, andmany patients die due to being unable to find a proper donor. Inaddition, finding a proper match is especially problematic forAfrican-Americans, Hispanics, Native Americans and people of mixedethnicity.

Hematopoietic stem cells (HSCs) are rare adult stem cells that have beenidentified in fetal bone marrow, fetal liver, aorta-gonad-mesonephros(AGM), umbilical cord blood (UCB), adult bone marrow, and peripheralblood, which are capable of differentiating into three cell lineagesincluding myeloerythroid (red blood cells, granulocytes, monocytes),megakaryocyte (platelets) and lymphoid (T-cells, B-cells, and naturalkiller) cells. HSCs, like bone marrow stem cells, are used in clinicaltransplantation protocols to treat a variety of diseases includingmalignant and non-malignant disorders.

Sources of HSCs include bone marrow and peripheral blood. To obtainmarrow cells, donors must undergo multiple aspirations to collectseveral thousand milliliters of bone marrow, a procedure that is carriedout under general anesthesia. To collect HSCs from the peripheral blood,the donor must be treated with granulocyte colony-stimulating factor toincrease the number of circulating HSCs. Both of these procedures entailsome risk and significant cost. Another source of HSCs is umbilical cordblood (UCB). UCB has major advantages over other sources of HSCs, suchas that from bone marrow and mobilized peripheral blood. Not only is UCBreadily available from many of the nearly 50 UCB banks across the UnitedStates, it also shows increased tolerance for mismatches with the hostmajor histocompatability complex (MEW). In addition to relativewidespread availability, these HSCs have several useful properties,including their decreased ability to induce immunological reactivity. Inmany cases, use of UCB incurs significantly less graft-versus-hostdisease compared to other sources of HSCs.

One barrier to the use of UCB is limited HSC numbers per cord atharvest. As cell dose has been shown to be a major determinant ofengraftment and survival after UCB transplantation, low stem cellnumbers represents the most significant barrier to successful UCB stemcell transplantation. The ability to expand ex vivo, prior totransplantation, the stem cell components of a single cord blood unitwill greatly increase the viability of this treatment modality. Infusingpatients with larger numbers of stem cells as opposed the limited cellsavailable in an unexpanded cord blood unit, should greatly increase thelikelihood of successful engraftment.

Expansion of HSCs has remained an important goal to develop advancedcell therapies for bone marrow transplantation and many blood disorders.Despite the identification of several hematopoietic growth factors, onlylimited expansion of HSCs has been observed. There thus remains a needfor new methods of expanding HSCs, particularly ex vivo methods.

BRIEF SUMMARY

In one aspect, a method of expanding a population of hematopoietic stemcells comprises contacting the population of hematopoietic stem cellswith an effective amount of an inhibitor of G-protein coupled receptor65 (GPR65) and providing a population of expanded, substantiallyundifferentiated hematopoietic stem cells. Also included is an expanded,substantially undifferentiated HSC population made by the foregoingprocess.

In another embodiment, a kit for expanding, ex vivo, the number ofhematopoietic stem cells in a population of hematopoietic stem cellscomprises an inhibitor of G-protein coupled receptor 65 (GPR65), andinstructions for the use of the inhibitor of G-protein coupled receptor65 (GPR65), wherein, when used, the kit provides expanded number ofhematopoietic stem cells.

In another aspect, a medium for carrying out ex vivo expansion ofhematopoietic stem cells in a population of hematopoietic stem cellscomprises a fluid medium suitable for maintaining viable stem cells, andan inhibitor of G-protein coupled receptor 65 (GPR65) present in themedium at a concentration suitable to provide expansion of thepopulation of HSCs.

In a still further aspect, a method for administering an expandedpopulation of hematopoietic stem cells to a patient in need thereof,comprises culturing a population of hematopoietic stem cells ex vivo ina hematopoietic stem cell expansion medium for a period of timesufficient to provide an expanded population of hematopoietic stemcells, wherein the hematopoietic stem cell expansion media comprises aninhibitor of G-protein coupled receptor 65 (GPR65), and administeringthe expanded population of hematopoietic stem cells to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-F show GATA-2 expression in +9.5^(−/−) AGM rescues CD31⁺c-KIT⁺hematopoietic and CD31⁺c-KIT⁻ endothelial cells. (A) AGM ex vivoretroviral infection and culture. (B) Flow cytometric analysis of GFP⁺cells within total live cells (6 litters: +9.5^(+/+)-Empty [n=8embryos]; +9.5^(−/−)-Empty [n=4 embryos]; +9.5^(−/−)-GATA-2 [n=6embryos]). (C) RT-PCR analysis of Gata2 mRNA levels in FACS-sortedCD31⁺c-KIT⁻ and CD31⁺c-KIT⁺ cells (6 litters: +9.5^(+/+)-Empty [n=8embryos]; +9.5^(−/−)-Empty [n=4 embryos]; +9.5^(−/−)-GATA-2 [n=6embryos]). (D) Representative plots from flow cytometric analysis ofCD31⁺c-KIT⁺ and CD31⁺c-KIT⁻ cell populations in infected AGMs after 96 hof ex vivo culture. (E and F) Quantitation of flow cytometry dataexpressed as the percentage of CD31⁺c-KIT⁻ and CD31⁺c-KIT⁺ cells in GPF⁺cells (E) and the percentage of GFP⁺ cells in CD31⁺c-KIT⁻ andCD31⁺c-KIT⁺ cells (F) (6 litters: +9.5^(+/+)-Empty [n=8 embryos];+9.5^(−/−) -Empty [n=4 embryos]; +9.5^(−/−)-GATA-2 [n=6 embryos]). Errorbars represent SEM. *, P<0.05; **, P<0.01; ***, P<0.001 (two-tailedunpaired Student's t-test).

FIGS. 2 A-D show rescued CD31⁺c-Kit⁺ cells exhibit normal colony-formingunit activity. (A and B) Quantitative analysis of colony-formingactivity of FACS-sorted CD31⁺c-KIT⁺ cells (6 litters: +9.5^(+/+)-Empty[n=9 embryos]; +9.5^(−/−)-Empty [n=7 embryos]; +9.5^(−/−)-GATA-2 [n=10embryos]). (C) Representative BFU-E, CFU-GM, and CFU-GEMM colonies fromFACS-sorted CD31⁺c-KIT⁺ cells. Scale bar, 2 mm. (D) Representativeimages of Wright-Giemsa stained cells obtained from colonies. Mac,macrophage; Ery, erythroblast; Neu, neutrophil; Mye, myeloid precursor.Scale Bar, 40 μm. Error bars represent SEM. *, P<0.05; **, P<0.01; ***,P<0.001 (two-tailed unpaired Student's t-test).

FIGS. 3 A-L show Global GPCR analysis in the AGM. (A) Schematic diagramshowing global GPCR analysis strategy. (B) A total of 314 non-olfactoryGPCRs were categorized into Secretin, adhesion, Glutamate,Frizzled/Taste2 and Rhodopsin families based on sequence homology. (C)Classification of 85 GPCRs expressed in the AGM (>5 transcripts permillion/TPM) into 5 families. (D) Bar graph depicting statisticallysignificant genes regulated by GATA-2 from RNA-seq analysis of+9.5^(+/+) and +9.5^(−/−) AGMs (Gao et al., 2013). Black bars indicategenes co-regulated by GATA-1 according to our prior microarray analysisof G1E-ER-GATA W/O β-estradiol treatment. (E) Expression pattern ofGata2, Adora3, Gpr65, Ltb4r1, and P2ry1 during erythropoiesis (P:Proerythroblast, B: Basophilic Erythroblast, O: PolyorthochromaticErythroblast, and R: Reticulocyte). (F) Time course of Gata2 and Gpr65expression following estradiol treatment in G1E-ER-GATA cells (n=3independent experiments). (G) RT-PCR analysis of Gata2 and Gpr65 inFACS-sorted R1, R2, R3, and R4/5 populations from fetal liver (n=3independent experiments). (H and I) ChIP signal map for Gpr65 in humanCD34 cells (H), mouse HPC7 cells, Lin⁻ bone marrow cells, and G1E cells(I). (J and K) RT-PCR analysis of Gata2 and Gpr65 mRNA in +9.5^(+/+) and+9.5^(−/−) AGM (5 litters: +9.5^(+/+)[n=8 embryos]; +9.5^(−/−) [n=6embryos]) and Yolk sac (3 litters: +9.5^(+/+)[n=7 embryos]; +9.5^(−/−)[n=5 embryos]) (J), and MAE cells expressing GATA-2 (K) (n=3 independentexperiments). (L) RNA-seq analysis of Gata2 and Gpr65 mRNA inFACS-sorted EC, HEC, HC, and HSCs from the AGM. EC: endothelial cells;HEC: hemogenic endothelial cells; HC: hematopoietic cell; HSC:hematopoietic stem cells. Error bars represent SEM. *, P<0.05(two-tailed unpaired Student's t-test).

FIGS. 4 A-G show Gpr65 suppresses hematopoiesis in the AGM andzebrafish. (A) Representative plots from flow cytometric analysis ofCD31⁺c-KIT⁺ and CD31⁺c-KIT⁺SCA1⁺ cell populations in control or Gpr65shRNA treated AGMs after 96 h of culture. (B) Quantitation of GFP⁺ cellswithin total live cells (9 litters: shLuc [n=22 embryos]; shGpr65 [n=26embryos]). (C) RT-PCR analysis of Gpr65 mRNA levels in FACS-sorted GFP⁺cells (6 litters: shLuc [n=15 embryos]; shGpr65 [n=15 embryos]). (D andE) Analysis of flow cytometry data expressed as the percentage ofCD31⁺c-KIT⁺ (D) and CD31⁺c-KIT⁺Scal1⁺ (E) cells in GFP⁺ cells (D: 9litters: shLuc [n=22 embryos]; shGpr65 [n=26 embryos]; E: 7 litters:shLuc [n=18]; shGpr65 [n=22]). (F) Representative images of ISH with theHSC markers Runx1/cMyb at 36 h post-fertilization. (G) Quantitation ofISH data expressed as percentage of embryos with high, medium, and lowRunx1/cMyb staining in total embryos (ATG MO 0 ng [124 embryos]; ATG MO4 ng [75 embryos]; ATG MO 6 ng [66 embryos]; SP MO 0 ng [97 embryos]; SPMO 4 ng [49 embryos]; SP MO 6 ng [58 embryos]). Gpr65_ATG MO: morpholinotargeting the translation start site of Gpr65; Gpr65 SP MO: morpholinoblocking the splicing of Gpr65. Error bars represent SEM. *, P<0.05;***, P<0.001 (two-tailed unpaired Student's t-test).

FIGS. 5 A-E show that psychosine promotes hematopoiesis in the AGM andGPR65 suppresses hematopoiesis by repressing Gata2 expression. (A)Representative flow cytometric plots of CD31⁺c-KIT⁺ and CD31⁺c-KIT⁺SCA1⁺cell populations in the AGM after 4 days of culture with 20 μMpsychosine. (B and C) The average percentage of CD31⁺c-KIT⁺ (B) andCD31⁺c-KIT⁺SCA1⁺ (C) cell populations with vehicle or psychosinetreatment (6 litters: control [n=19 embryos]; psychosine [n=20embryos]). (D and E) RT-PCR analysis of Gpr65, Gata2, and Runx1 mRNA (D)and Gata2 primary transcript (E) in FACS-sorted CD31⁺c-KIT⁻ cells (n=3independent experiments). Error bars represent SEM. *, P<0.05; **,P<0.01; ***, P<0.001 (two-tailed unpaired Student's t-test).

FIGS. 6 A-L show that GPR65 enhances H4K20me1 and limits Scl/TAL1occupancy at the +9.5 enhancer. (A) Representative flow cytometric plotsof erythroid maturation based on expression of CD71 and Ter119 after 3days of HSPC expansion. The average percentage of total cells in R1through R5 populations after treatment with control or Gpr65 shRNA isdepicted on the right (n=3 independent experiments). (B) RT-PCR analysisof Gpr65 mRNA in fetal liver cells (n=5 independent experiments) (left).Western blot analysis of GPR65 in fetal liver cells (top right) andquantification of the GPR65 and α-tubulin intensities by densitometry(n=8 independent experiments) (bottom right). (C) RT-PCR analysis ofGata2 mRNA and Gata2 primary transcript in total fetal liver cells (n=5independent experiments). (D) Western blot analysis of GATA-2 in fetalliver cells (top) and quantification of GATA-2/α-tubulin ratio bydensitometry (n=6 independent experiments) (bottom). (E) RT-PCR analysisof GATA-2 target gene expression in fetal liver cells (n=5 independentexperiments). (F, G, H, and I) Real-time RT-PCR analysis of Gpr65 mRNA(F), Gata2 mRNA (G), Gata2 primary transcript (G) and GATA-2 targetgenes in FACS-sorted R2 cells (n=3 independent experiments) (I). Westernblot analysis of GATA-2 in FACS-sorted R2 cells and quantification ofthe intensities of the GATA-2 to α-tubulin band by densitometricanalysis (n=4 independent experiments) (H). (J) Strategy in which fetalliver HSPCs were isolated from E14.5 embryos heterozygous for the +9.5site at Gata2. Cells were infected with Control or shGpr65 and culturedunder expansion conditions for 3 days (left). Allele-specific real-timeRT-PCR analysis of Gata2 transcripts from the WT and mutant +9.5 allelesin total fetal liver cells (n=3 independent experiments) (middle) andFACS-sorted R2 cells (n=3 independent experiments) (right). (K) RT-PCRanalysis of Setd8 mRNA in fetal liver cells treated with control orGpr65 shRNA (n=5 independent experiments) (top left). H4K20me1, GATA-1and Scl/TAL1 chromatin occupancy measured by quantitative CMP at the+9.5 site in fetal liver cells cultured under expansion medium andtreated with control or Gpr65 shRNA (H4K20me1: n=8 independentexperiments; GATA-1: n=4 independent experiments; Scl/TAL1: n=6independent experiments) (right). H4K20me1 levels at the repressed MyoDpromoter and at the active Eif3k promoter serve as controls (middleleft). Scl/TAL1 chromatin occupancy at 114 kb upstream from the c-Kitpromoter and Lyl1 exon1 as controls (bottom left). The dashed lineillustrates the highest value obtained with PI antibody. (L) Model:GATA-2, a positive regulator of hematopoiesis, upregulates Gpr65, whichencodes a negative regulator of hematopoiesis, to control HSC emergence.GPR65 represses Gata2 expression by increasing H4K20me1, a repressivechromatin mark, which in turn restricts +9.5 occupancy by the activatorScl/TAL1. Error bars represent SEM. *, P<0.05; **, P<0.01; ***, P<0.001(two-tailed unpaired Student's t-test).

FIG. 7 A-D show that psychosine increased Gata2 expression and slightlyblocks erythroipoiesis. (A) Representative flow cytometric plots oferythroid maturation based on CD71 and Ter119 expression 3 days afterHSPC expansion (left). The average percentage of cells in R1 through R5populations after treatment with control (DMSO) or psychosine (right).(B) RT-PCR analysis of Gata2 mRNA and Gata2 primary transcript in totalfetal liver cells. (C) RT-PCR analysis of Gata2 mRNA and Gata2 primarytranscript in FACS-sorted R2 cells. (D) Western blot of GATA-2 andα-Tubulin in FACS-sorted R2 cells. Error bars represent SEM. *P<0.05;**P<0.01 (two-tailed unpaired Student's t test)

FIG. 8 shows the structure of sphingosine, galactosylsphingosine(psychosine), glucosylsphingosine, and lactosylsphingosine.

FIG. 9 A-D show that glucosylsphingosine, but not lactosylsphingosineshowed similar activity as psychosine. (A) Representative flowcytometric plots of erythroid maturation based on CD71 and Ter119expression 3 days after HSPC expansion. (B) The average percentage ofcells in R1 through R5 populations after infection with psychosine(GalSph), glucosylsphingosine (GluSph), or lactosylsphingosine (LacSph).(C) RT-PCR analysis of Gata2 mRNA in fetal liver cells. (D) Western blotanalysis of GATA-2 in fetal liver cells (left), and quantification ofGATA-2 and tubulin intensities by densitometry (right). Error barsrepresent SEM. *P<0.05; **P<0.01; ***P<0.001 (two-tailed unpairedStudent's t test)

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Described herein are novel methods of expanding hematopoietic stem cells(HSCs). The inventors of the present application have unexpectedly foundthat GATA-2, a positive regulator of hematopoiesis, upregulated Gpr65,which encodes a negative regulator of hematopoiesis. Thus, inhibitors ofG-protein coupled receptor 65 (GPR65) can be used to provide populationsof expanded, substantially undifferentiated hematopoietic stem cells.Such expanded hematopoietic stem cell populations are critical for thedevelopment of advanced cell therapies for bone marrow transplantationand many blood disorders.

Establishment and maintenance of the adult hematopoietic system requiresHSCs generated from a unique type of endothelial cell (hemogenic) in theaorta-gonad-mesonephros (AGM) region of the mammalian embryo. HSCsdevelop in cell clusters that bud off from hemogenic endothelium, aprocess termed endothelial to hematopoietic transition (EHT). The HSCsmigrate to and colonize the fetal liver and subsequently the bonemarrow.

Many questions remain unanswered regarding the molecular constituentsand mechanistic steps controlling EHT. Considerable efforts have focusedon defining the relevant regulatory proteins and networks. An emergingtheme is that master regulatory transcription factors co-localize atcis-elements of target genes and establish complex genetic networks thatcontrol hematopoiesis. Whether this combinatorial mechanism operates inhemogenic endothelium is unclear, and the relationship betweenmechanisms controlling EHT and those conferring HSC multi-potency is notunderstood. A shared component of the mechanisms involves thetranscription factor GATA-2, which is essential for definitivehematopoiesis. GATA-2 functions in hemogenic endothelium to induce EHTand promotes HSC function.

Evidence for GATA-2 control of EHT emerged from analysis of mutant micelacking a Gata2 intronic cis-regulatory element (+9.5) and fromconditional Gata2 knockout mice. GATA-2 occupies sites-77, -3.9, -2.8,-1.8 upstream and an intronic site +9.5 kb downstream of the Gata2 1Spromoter, and GATA-1 replacement of GATA-2 at these sites is tightlylinked to Gata2 repression during erythropoiesis. Unlike deletions ofthe other GATA-2 occupancy sites, the +9.5 site deletion uniquelydecreases Gata2 expression in AGM hemogenic endothelium, deregulatesgenes encoding positive regulators of hematopoiesis, and abrogateshemogenic endothelial activity to generate HSCs. The defective HSCgenerator of +9.5^(−/−) embryos causes a severe HSPC deficiency in thefetal liver and embryonic lethality at E13-14.

GATA-2 regulates a large gene cohort in hemogenic endothelium, and theindividual gene constituents do not parse into a predominant molecularpathway. These constituents include Runx1, Lyl1, and Mpl, positivemediators of HSC generation and/or function. The identification ofconstituents of the GATA-2-dependent genetic network bearing theseattributes will reveal how GATA-2 triggers endothelium to form a stemcell. Moreover, as GATA-2 lacks ligand binding and catalytic sites,features that can be leveraged for small molecule/drug binding, thesemechanistic insights will usher in strategies to promote HSCgeneration/function for transplantation and to inhibit proliferation orsurvival of HSPC-derived leukemia cells.

The most common targets for FDA-approved drugs are the GPCRs. The GPCRfamily consists of 341 non-olfactory receptors that are classified asrhodopsin, secretin, glutamate, adhesion and Frizzed/Taste2 based onsequence homology. CXCR4, a rhodopsin-like GPCR, has a critical role inHSPC survival, proliferation, migration and engraftment. CXCR4antagonists are used as mobilizing agents for stem cell transplantation.Previously, the present inventors described +9.5 site-mediatedupregulation of Gpr56 expression, and a loss-of-function analysisindicated that GPR56 promotes HSC emergence in the AGM. However,analyses of the role of Gpr56 in HSC maintenance and function yieldedopposite conclusions—either it had no role or it promoted HSCmaintenance.

By systematically profiling expression of the entire GPCR cohort in theAGM, the present inventors have now discovered a small subset ofGATA-2-regulated GPCRs, a smaller cohort that are GATA-2- andGATA-1-regulated, and one in particular, GPR65, that suppresses AGMhematopoiesis. Surprisingly, GATA-2, a positive regulator ofhematopoiesis, upregulated Gpr65, which encodes a negative regulator ofhematopoiesis. These results provide evidence for a GATA factor-GPCRincoherent type I feedforward loop as a vital component of theGATA-2-dependent genetic network that controls HSC emergence.

In one aspect, a method of expanding a population of hematopoietic stemcells comprises contacting the population of hematopoietic stem cellswith an effective amount of an inhibitor of G-protein coupled receptor65 (GPR65) and providing a population of expanded, substantiallyundifferentiated hematopoietic stem cells. For example, the methodcomprises culturing the population of HSCs in an HSC expansion mediumfor a period of time sufficient to expand the number of HSCs in the HSCpopulation, wherein the HSC expansion media comprises an inhibitor ofG-protein coupled receptor 65 (GPR65).

As used herein, “expand”, “expanding” and like terms means to increasethe number of undifferentiated HSCs in the population relative to thenumber of HSCs in the original population in vitro, in vivo or ex vivousing any of the methods disclosed herein. The expansion of the HSCs canbe evaluated by a cell marker analysis (for example, counting the cellscorresponding to CD34⁺ by FACS), quantitative analysis based on thecolony assay method, and the like. In one aspect, expanding is ex vivoand the number of undifferentiated cells in the population of HSCs isincreased by greater than 1.25-fold. In one aspect, the population ofHSCs is enriched to a clinically relevant number. In one aspect, thepopulation of HSCs is enriched for CD34⁺ cells, meaning that thepercentage of CD34⁺ cells relative to other cells in a population isincreased. Additional stem cell markers include CD38⁻, DR⁻, CD45⁺,CD90⁺, CD117⁺, CD123⁺, and CD133⁺.

In certain aspect, the expanded HSC population has “long term,multi-lineage repopulating potential” meaning that the expanded HSCs arecapable of repopulating many different types of blood cells inirradiated recipients upon transplantation and/or cells that possesshigh proliferative potential in vitro.

An HSC population is “substantially undifferentiated” if a sufficientnumber of cells in that population retain the ability to self-renew andcan give rise to various differentiated cell types when transplantedinto a recipient, for example, in the case of an HSC population,repopulating the HSC lineage when transplanted. As used herein, “withoutsignificant differentiation” means the expanded stem cell population hasa sufficient number of cells that maintain a multi-lineagedifferentiation potential that the full scope of a target stem lineagemay be regenerated upon transplantation of the expanded stem cellpopulation into a recipient. For example, the expanded HSC population,when transplanted into a recipient, is capable of regenerating theentire hematopoietic cell lineage.

In an embodiment, the population of HSCs is obtained from a mammaliantissue selected from umbilical cord blood, peripheral blood, and bonemarrow. HSCs may be the subject's own cells (autologous) or those of adonor (allogeneic). In a more specific embodiment, the population ofHSCs is obtained from mammalian cord blood. In certain aspects, the HSCsare of human origin.

As used herein, “obtained” from a tissue means a conventional method ofharvesting or partitioning tissue from a donor. For example, the tissuemay be obtained from a blood sample, such as a peripheral or cord bloodsample, or harvested from bone marrow. Methods for obtaining suchsamples are well known to one of ordinary skill in the art. The samplesmay be fresh, i.e., obtained from the donor without freezing. Moreover,the samples may be further manipulated to remove extraneous or unwantedcomponents prior to expansion. The samples may also be obtained from apreserved stock. For example, in the case of peripheral or cord blood,the samples may be withdrawn from a cryogenically or otherwise preservedbank of such blood. Donor animals are mammals including a primate, suchas a human; or laboratory animals such as mice, rats, dogs, and pigs.The sample may be obtained from an autologous or allogeneic donor orsource. In certain aspects, the sample is obtained from an autologoussource. Methods for isolation of hematopoietic stem cells may involvesubsequent purification techniques based on cell surface markers andfunctional characteristics.

In another aspect, included herein is an expanded, substantiallyundifferentiated HSC population made by contacting a population ofhematopoietic stem cells with an effective amount of an inhibitor ofG-protein coupled receptor 65 (GPR65) to provide the population ofexpanded, substantially undifferentiated hematopoietic stem cells.

Further included herein is a kit for expanding, ex vivo, the number ofhematopoietic stem cells (HSC) in a population of HSCs, the kitcomprising an inhibitor of G-protein coupled receptor 65 (GPR65), andinstructions for the use of the inhibitor of G-protein coupled receptor65 (GPR65), wherein, when used, the kit provides expanded number ofHSCs. Exemplary instructions for use include, for example, a detaileddescription of the kit components, reaction times/temperature forexpanding HSCs, and the like.

In another aspect, a medium for carrying out ex vivo expansion of HSCsin a population of HSCs comprises a fluid medium suitable formaintaining viable stem cells, and an inhibitor of G-protein coupledreceptor 65 (GPR65) present in the medium at a concentration suitable toprovide expansion of the population of HSCs. An “HSC expansion medium”is a medium suitable for expanding the number of HSCs in a culture andincludes, for example, StemPro® 34 medium, GE HyClone® medium, Stemline®HSC expansion medium from Sigma-Aldrich, and StemSpan™ SFEM II fromSTEMCELL Technologies. The medium comprises e.g., 20 μm of an inhibitorof G-protein coupled receptor 65 (GPR65) for example.

A further embodiment includes a method for administering an expandedpopulation of hematopoietic stem cells to a patient in need thereof,comprising culturing a population of hematopoietic stem cells ex vivo ina hematopoietic stem cell expansion medium for a period of timesufficient to provide an expanded population of hematopoietic stemcells, wherein the hematopoietic stem cell expansion medium comprises aninhibitor of G-protein coupled receptor 65 (GPR65), and administeringthe expanded population of hematopoietic stem cells to the patient.Exemplary patients include patients in need of treatment for leukemia,multiple myeloma, some types of lymphoma, graft versus host disease, andgenetic disorders of the blood and immune system including aplasticanemia, sickle cell anemia, Severe Combined Immune Deficiency (SCID),Wiskott-Aldrich Syndrome (WAS), IPEX Syndrome, HemophagocyticLymphohistiocytosis (HLH), X-linked Lymphoproliferative Disease (XLP)and Chronic Granulomatous Disease (CGD).

In one aspect, the method provides expanded HSCs that, upon transplantinto a recipient, exhibit greater than 5% donor repopulation, such asgreater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%,80%, 85%, or 90% donor repopulation. Specifically, the method providesexpanded HSCs that, upon transplant into a recipient, exhibit greaterthan 25%, 35%, 45%, or 60% donor repopulation. An exemplary recipient isa mammal, for example, a primate, such as a human; or laboratory animalssuch as mice, rats, dogs, and pigs. The term “recipient” is usedinterchangeably with “patient.”

In one aspect, the inhibitor of G-protein coupled receptor 65 (GPR65) isa glucosphingolipid or a galactosphingolipid as well as their salts andderivatives. Exemplary compounds include psychosine(galactosphingolipid), dihydropsychosine, lysosulfatide(sulfogalactosylsphingosine), and glucosylsphingosine, and the like.

In another aspect, the inhibitor of G-protein coupled receptor 65(GPR65) is an antibody that both binds to and inhibits G-protein coupledreceptor 65 (GPR65).

In one aspect, the GPR65 inhibitor is an inhibitory nucleic acidmolecule, wherein administration of the inhibitory nucleic acid moleculeselectively decreases the expression of GPR65. The term “inhibitorynucleic acid molecule” means a single stranded, double-stranded ortriple-stranded RNA or DNA, specifically RNA, such as triplexoligonucleotides, ribozymes, aptamers, small interfering RNA includingsiRNA (short interfering RNA) and shRNA (short hairpin RNA), antisenseRNA, or a portion thereof, or an analog or mimetic thereof, that iscapable of reducing or inhibiting the expression of a target gene orsequence. Inhibitory nucleic acids can act by, for example, mediatingthe degradation or inhibiting the translation of mRNAs which arecomplementary to the interfering RNA sequence. An inhibitory nucleicacid, when administered to a mammalian cell, results in a decrease(e.g., by 5%, 10%, 25%, 50%, 75%, or even 90-100%) in the expression(e.g., transcription or translation) of a target sequence. Typically, anucleic acid inhibitor comprises or corresponds to at least a portion ofa target nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. Inhibitory nucleic acids may have substantial or completeidentity to the target gene or sequence, or may include a region ofmismatch (i.e., a mismatch motif). The sequence of the inhibitorynucleic acid can correspond to the full-length target gene, or asubsequence thereof. In one aspect, the inhibitory nucleic acidmolecules are chemically synthesized.

The specific sequence utilized in design of the inhibitory nucleic acidsis a contiguous sequence of nucleotides contained within the expressedgene message of the target. Factors that govern a target site for theinhibitory nucleic acid sequence include the length of the nucleic acid,binding affinity, and accessibility of the target sequence. Sequencesmay be screened in vitro for potency of their inhibitory activity bymeasuring inhibition of target protein translation and target relatedphenotype, e.g., inhibition of cell proliferation in cells in culture.In general it is known that most regions of the RNA (5′ and 3′untranslated regions, AUG initiation, coding, splice junctions andintrons) can be targeted using antisense oligonucleotides. Programs andalgorithms, known in the art, may be used to select appropriate targetsequences. In addition, optimal sequences may be selected utilizingprograms designed to predict the secondary structure of a specifiedsingle stranded nucleic acid sequence and allowing selection of thosesequences likely to occur in exposed single stranded regions of a foldedmRNA. Methods and compositions for designing appropriateoligonucleotides may be found, for example, in U.S. Pat. No. 6,251,588,the contents of which are incorporated herein by reference.

Phosphorothioate antisense oligonucleotides may be used. Modificationsof the phosphodiester linkage as well as of the heterocycle or the sugarmay provide an increase in efficiency. Phosphorothioate is used tomodify the phosphodiester linkage. An N3′-P5′ phosphoramidate linkagehas been described as stabilizing oligonucleotides to nucleases andincreasing the binding to RNA. A peptide nucleic acid (PNA) linkage is acomplete replacement of the ribose and phosphodiester backbone and isstable to nucleases, increases the binding affinity to RNA, and does notallow cleavage by RNAse H. Its basic structure is also amenable tomodifications that may allow its optimization as an antisense component.With respect to modifications of the heterocycle, certain heterocyclemodifications have proven to augment antisense effects withoutinterfering with RNAse H activity. An example of such modification isC-5 thiazole modification. Finally, modification of the sugar may alsobe considered. 2′-O-propyl and 2′-methoxyethoxy ribose modificationsstabilize oligonucleotides to nucleases in cell culture and in vivo.

Short interfering (si) RNA technology (also known as RNAi) generallyinvolves degradation of an mRNA of a particular sequence induced bydouble-stranded RNA (dsRNA) that is homologous to that sequence, thereby“interfering” with expression of the corresponding gene. A selected genemay be repressed by introducing a dsRNA which corresponds to all or asubstantial part of the mRNA for that gene. Without being held totheory, it is believed that when a long dsRNA is expressed, it isinitially processed by a ribonuclease III into shorter dsRNAoligonucleotides of as few as 21 to 22 base pairs in length.Accordingly, siRNA may be effected by introduction or expression ofrelatively short homologous dsRNAs. Exemplary siRNAs have sense andantisense strands of about 21 nucleotides that form approximately 19nucleotides of double stranded RNA with overhangs of two nucleotides ateach 3′ end.

siRNA has proven to be an effective means of decreasing gene expressionin a variety of cell types. siRNA typically decreases expression of agene to lower levels than that achieved using antisense techniques, andfrequently eliminates expression entirely. In mammalian cells, siRNAsare effective at concentrations that are several orders of magnitudebelow the concentrations typically used in antisense experiments.

The double stranded oligonucleotides used to effect RNAi arespecifically less than 30 base pairs in length, for example, about 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, or 17 base pairs or less inlength, and contain a segment sufficiently complementary to the targetmRNA to allow hybridization to the target mRNA. Optionally, the dsRNAoligonucleotide includes 3′ overhang ends. Exemplary 2-nucleotide 3′overhangs are composed of ribonucleotide residues of any type and may becomposed of 2′-deoxythymidine residues, which lowers the cost of RNAsynthesis and may enhance nuclease resistance of siRNAs in the cellculture medium and within transfected cells. Exemplary dsRNAs aresynthesized chemically or produced in vitro or in vivo using appropriateexpression vectors. Longer RNAs may be transcribed from promoters, suchas T7 RNA polymerase promoters, known in the art.

Longer dsRNAs of 50, 75, 100, or even 500 base pairs or more also may beutilized in certain embodiments. Exemplary concentrations of dsRNAs foreffecting RNAi are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5 nM, 25 nM,or 100 nM, although other concentrations may be utilized depending uponthe nature of the cells treated, the gene target and other factorsreadily identified by one of ordinary skill in the art.

Compared to siRNA, shRNA offers advantages in silencing longevity anddelivery options. Vectors that produce shRNAs, which are processedintracellularly into short duplex RNAs having siRNA-like propertiesprovide a renewable source of a gene-silencing reagent that can mediatepersistent gene silencing after stable integration of the vector intothe host-cell genome. Furthermore, the core silencing ‘hairpin’ cassettecan be readily inserted into retroviral, lentiviral, or adenoviralvectors, facilitating delivery of shRNAs into a broad range of celltypes.

A hairpin can be organized in either a left-handed hairpin (i.e.,5′-antisense-loop-sense-3′) or a right-handed hairpin (i.e.,5′-sense-loop-antisense-3′). The shRNA may also contain overhangs ateither the 5′ or 3′ end of either the sense strand or the antisensestrand, depending upon the organization of the hairpin. If there are anyoverhangs, they are specifically on the 3′ end of the hairpin andinclude 1 to 6 bases. The overhangs can be unmodified, or can containone or more specificity or stabilizing modifications, such as a halogenor O-alkyl modification of the 2′ position, or internucleotidemodifications such as phosphorothioate, phosphorodithioate, ormethylphosphonate modifications. The overhangs can be ribonucleic acid,deoxyribonucleic acid, or a combination of ribonucleic acid anddeoxyribonucleic acid.

Additionally, a hairpin can further comprise a phosphate group on the5′-most nucleotide. The phosphorylation of the 5′-most nucleotide refersto the presence of one or more phosphate groups attached to the 5′carbon of the sugar moiety of the 5′-terminal nucleotide. Specifically,there is only one phosphate group on the 5′ end of the region that willform the antisense strand following Dicer processing. In one exemplaryembodiment, a right-handed hairpin can include a 5′ end (i.e., the free5′ end of the sense region) that does not have a 5′ phosphate group, orcan have the 5′ carbon of the free 5′-most nucleotide of the senseregion being modified in such a way that prevents phosphorylation. Thiscan be achieved by a variety of methods including, but not limited to,addition of a phosphorylation blocking group (e.g., a 5′-O-alkyl group),or elimination of the 5′-OH functional group (e.g., the 5′-mostnucleotide is a 5′-deoxy nucleotide). In cases where the hairpin is aleft-handed hairpin, preferably the 5′ carbon position of the 5′-mostnucleotide is phosphorylated.

Hairpins that have stem lengths longer than 26 base pairs can beprocessed by Dicer such that some portions are not part of the resultingsiRNA that facilitates mRNA degradation. Accordingly the first region,which may include sense nucleotides, and the second region, which mayinclude antisense nucleotides, may also contain a stretch of nucleotidesthat are complementary (or at least substantially complementary to eachother), but are or are not the same as or complementary to the targetmRNA. While the stem of the shRNA can include complementary or partiallycomplementary antisense and sense strands exclusive of overhangs, theshRNA can also include the following: (1) the portion of the moleculethat is distal to the eventual Dicer cut site contains a region that issubstantially complementary/homologous to the target mRNA; and (2) theregion of the stem that is proximal to the Dicer cut site (i.e., theregion adjacent to the loop) is unrelated or only partially related(e.g., complementary/homologous) to the target mRNA. The nucleotidecontent of this second region can be chosen based on a number ofparameters including but not limited to thermodynamic traits orprofiles.

Modified shRNAs can retain the modifications in the post-Dicer processedduplex. In exemplary embodiments, in cases in which the hairpin is aright handed hairpin (e.g., 5′-S-loop-AS-3′) containing 2-6 nucleotideoverhangs on the 3′ end of the molecule, 2′-O-methyl modifications canbe added to nucleotides at position 2, positions 1 and 2, or positions1, 2, and 3 at the 5′ end of the hairpin. Also, Dicer processing ofhairpins with this configuration can retain the 5′ end of the sensestrand intact, thus preserving the pattern of chemical modification inthe post-Dicer processed duplex. Presence of a 3′ overhang in thisconfiguration can be particularly advantageous since blunt endedmolecules containing the prescribed modification pattern can be furtherprocessed by Dicer in such a way that the nucleotides carrying the 2′modifications are removed. In cases where the 3′ overhang ispresent/retained, the resulting duplex carrying the sense-modifiednucleotides can have highly favorable traits with respect to silencingspecificity and functionality. Examples of exemplary modificationpatterns are described in detail in U.S. Patent Publication No.20050223427 and International Patent Publication Nos. WO 2004/090105 andWO 2005/078094, the disclosures of each of which are incorporated byreference herein in their entirety.

shRNA may comprise sequences that were selected at random, or accordingto a rational design selection procedure. For example, rational designalgorithms are described in International Patent Publication No. WO2004/045543 and U.S. Patent Publication No. 20050255487, the disclosuresof which are incorporated herein by reference in their entireties.Additionally, it may be desirable to select sequences in whole or inpart based on average internal stability profiles (“AISPs”) or regionalinternal stability profiles (“RISPs”) that may facilitate access orprocessing by cellular machinery.

Ribozymes are enzymatic RNA molecules capable of catalyzing specificcleavage of mRNA, thus preventing translation. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by an endonucleolytic cleavageevent. The ribozyme molecules specifically include (1) one or moresequences complementary to a target mRNA, and (2) the well-knowncatalytic sequence responsible for mRNA cleavage or a functionallyequivalent sequence (see, e.g., U.S. Pat. No. 5,093,246, which isincorporated herein by reference in its entirety).

While ribozymes that cleave mRNA at site-specific recognition sequencescan be used to destroy target mRNAs, hammerhead ribozymes mayalternatively be used. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. Specifically, the target mRNA has the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully in U.S.Pat. No. 5,633,133, the contents of which are incorporated herein byreference.

Gene targeting ribozymes may contain a hybridizing region complementaryto two regions of a target mRNA, each of which is at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides(but which need not both be the same length).

Hammerhead ribozyme sequences can be embedded in a stable RNA such as atransfer RNA (tRNA) to increase cleavage efficiency in vivo. Inparticular, RNA polymerase III-mediated expression of tRNA fusionribozymes is well known in the art. There are typically a number ofpotential hammerhead ribozyme cleavage sites within a given target cDNAsequence. Specifically, the ribozyme is engineered so that the cleavagerecognition site is located near the 5′ end of the target mRNA—toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts. Furthermore, the use of any cleavagerecognition site located in the target sequence encoding differentportions of the target mRNA would allow the selective targeting of oneor the other target genes.

Ribozymes also include RNA endoribonucleases (“Cech-type ribozymes”)such as the one which occurs naturally in Tetrahymena thermophile,described in International Patent Publication No. WO 88/04300. TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence where after cleavage of the target RNA takesplace. In one embodiment, Cech-type ribozymes target eight base-pairactive site sequences that are present in a target gene or nucleic acidsequence.

Ribozymes can be composed of modified oligonucleotides (e.g., forimproved stability, targeting, etc.) and can be chemically synthesizedor produced through an expression vector. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency. Additionally, in certain embodiments, aribozyme may be designed by first identifying a sequence portionsufficient to cause effective knockdown by RNAi. Portions of the samesequence may then be incorporated into a ribozyme.

Alternatively, target gene expression can be reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe gene (i.e., the promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the gene in target cells in thebody. Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription are specifically single stranded andcomposed of deoxyribonucleotides. The base composition of theseoligonucleotides should promote triple helix formation via Hoogsteenbase pairing rules, which generally require sizable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the target sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Inhibitory nucleic acids can be administered directly or delivered tocells by transformation or transfection via a vector, including viralvectors or plasmids, into which has been placed DNA encoding theinhibitory oligonucleotide with the appropriate regulatory sequences,including a promoter, to result in expression of the inhibitoryoligonucleotide in the desired cell. Known methods include standardtransient transfection, stable transfection and delivery using virusesranging from retroviruses to adenoviruses. Delivery of nucleic acidinhibitors by replicating or replication-deficient vectors iscontemplated. Expression can also be driven by either constitutive orinducible promoter systems. In other embodiments, expression may beunder the control of tissue or development-specific promoters.

Vectors may be introduced by transfection using carrier compositionssuch as Lipofectamine 2000 (Life Technologies) or Oligofectamine™ (LifeTechnologies). Transfection efficiency may be checked using fluorescencemicroscopy for mammalian cell lines after co-transfection ofhGFP-encoding pAD3.

The effectiveness of the inhibitory oligonucleotide may be assessed byany of a number of assays, including reverse transcriptase polymerasechain reaction or Northern blot analysis to determine the level ofexisting human sclerostin mRNA, or Western blot analysis usingantibodies which recognize the human sclerostin protein, aftersufficient time for turnover of the endogenous pool after new proteinsynthesis is repressed.

As used herein, the term substantially complementary means that thecomplement of one molecule is substantially identical to the othermolecule. Two nucleic acid or protein sequences are consideredsubstantially identical if, when optimally aligned, they share at leastabout 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical. Optimal alignment of sequences forcomparisons of identity may be conducted using a variety of algorithmsknown in the art. Sequence identity may also be determined using theBLAST algorithm.

Also included herein is a composition comprising a small interferingRNA, the small interfering RNA comprising 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides ofGpr65 (NM_008152; SEQ ID NO: 1). Exemplary small interfering RNAs aresiRNA and shRNA.

Further included are pharmaceutical compositions comprising apharmaceutically acceptable excipient and a small interfering RNA, thesmall interfering RNA comprising 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides of SEQID NO: 1. Exemplary shRNAs include:

miR-30-context Gpr65 shRNA sequences: (SEQ ID NO: 2)TGCTGTTGACAGTGAGCGAGCAGGTTAAGTTACATGGTATTAGTGAAGCCACAGATGTAATACCATGTAACTTAACCTGCCTGCCTACTGCCTCGGA (SEQ ID NO: 3)TGCTGTTGACAGTGAGCGAAAAGATGAAACGAGTGTTGAATAGTGAAGCCACAGATGTATTCAACACTCGTTTCATCTTTCTGCCTACTGCCTCGGA

In one aspect, the GPR65 inhibitor comprises a small interfering RNA anda glucosphingolipid or a galactosphingolipid.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES

Methods

Mice and embryo generation. Gata2 +9.5^(−/+) mice generated viahomologous recombination in ES cells are described in the art. Stagedembryos were obtained from timed mating between Gata2 +9.5heterozygotes. The detection of the vaginal plug was considered as day0.5. Pregnant females were euthanized with CO₂, and fresh embryos weretransferred into cold PBS for dissection. Mice and embryos are genotypedby PCR. All animal experiments were carried out with the ethicalapproval of the AAALAC International (Association for the Assessment andAccreditation of Laboratory Animal Care) at the University ofWisconsin-Madison.

AGM explant culture. AGMs were dissected from E11.5 embryos. Freshlyisolated AGMs were infected as below and cultured as described. Inbrief, intact AGMs were cultured for 4 days on Durapore® filters(Millipore) at the air-liquid interface in IMDM⁺ Media (Iscove'smodified Dulbecco's media [Gibco] supplemented with 20% fetal bovineserum [FBS; Gemini], L-glutamine [Gibco; 4 mM], 1%penicillin/streptomycin [Cellgro], mercaptoethanol [0.1 mM], IL-3 [R&D;100 ng/ml], Flt3L [R&D; 100 ng/ml], and 1.5% conditioned medium from aKit ligand-producing CHO cell line).

Primary erythroid precursor cell isolation. Primary erythroid precursorswere isolated from E14.5 fetal livers using the EasySep™ negativeselection Mouse Hematopoietic Progenitor Cell Enrichment Kit (StemCellTechnologies). Briefly, fetal livers were dissociated by pipetting andresuspended at 5×10⁷ cell/ml in PBS containing 2% FBS, 2.5 mM EDTA, and10 mM glucose. EasySep™ Mouse Hematopoietic Progenitor Cell EnrichmentCocktail was added at 50 μg/ml supplemented with 2.5 μg/mlbiotin-conjugated CD71 antibody (eBioscience). After 15 min incubationon ice, the cells were washed by centrifugation for min at 1200 rpm at4° C. and resuspended at 5×10⁷ cell/ml in PBS containing 2% FBS, 2.5 mMEDTA, and 10 mM glucose, and EasySep™ Biotin Selection Cocktail wasadded at 100 μg/ml. After 15 min incubation at 4° C., EasySep™ MouseProgenitor Magnetic Microparticles were added at 50 μg/ml. After 10 minincubation at 4° C., cells were resuspended to 2.5 ml and incubated witha magnet for 3 min. Unbound cells were carefully transferred into 15 mltube and used for subsequent experiments.

Cell culture. G1E-ER-GATA-1 cells were cultured with or without 1 μMestradiol as previously described. Fetal liver erythroid precursor cellswere cultured and maintained at a density of 2.5×10⁵-1×10⁶ cells/ml inStemPro®-34 (Gibco) supplemented with 10% nutrient supplement (Gibco), 2mM L-glutamine (Cellgro), 1% Penicillin/streptomycin (Cellgro), 100 μMmonothioglycerol (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich),0.5 U/ml of erythropoietin, and 1% conditioned medium from a kitligand-producing CHO cell line for expansion. Cells were cultured in ahumidified incubator at 37° C. (5% carbon dioxide).

Retroviral infection. MiR-30-context Gpr65 shRNA were cloned intoMSCV-PIG (IRES-GFP) vector (kindly provided by Dr. Mitchell Weiss) usingBgl II and Xho I restriction sites. Retrovirus expressing Gata2,containing a Gata2 cDNA, an empty vector, shRNA targeting luciferase, orGpr65 were produced by co-transfecting 293T cells with pCL-Eco packagingvector. Retroviral supernatant was collected 24 h and 48 hpost-transfection and centrifuged to remove cells and debris. TheRetro-X™ Concentrator from Clontech (Cat. No. 631455) was used forconcentrating retrovirus to get high-concentrated retrovirus. Briefly,the retroviral supernatant was mixed with the Retro-X™ Concentrator androtated at 4° C. for 1 h. The mixture was then centrifuged at 1,500 gfor 45 minutes to obtain a high-titer virus-containing pellet which canbe easily resuspended in 1/100^(th) of the original volume using IMDMMedia (Iscove's modified Dulbecco's media) [Gibco] supplemented with 20%FBS [Gemini] and 1% penicillin/streptomycin [Cellgro].

Freshly isolated AGMs from E11.5 embryos were infected with 20 μl ofhigh-concentrated retrovirus and 8 μg/ml polybrene in 500 μl of AGMexplant culture media at 2800 rpm for 90 min at 30° C. Aftercentrifugation, AGMs were subjected for AGM explant culture ex vivo.

Freshly isolated primary erythroid precursor cells were spinfected with100 ul of retrovirus supernatant and 8 μg/ml polybrene in 400 μl offetal liver expansion media at 2800 RPM for 90 min at 30° C. Aftercentrifugation, 500 μl pre-warmed fetal liver expansion medium wasadded, and cells were incubated at 37° C. for 72 h.

miR-30-context Gpr65 shRNA sequence: (SEQ ID NO: 2)TGCTGTTGACAGTGAGCGAGCAGGTTAAGTTACATGGTATTAGTGAAGCCACAGATGTAATACCATGTAACTTAACCTGCCTGCCTACTGCCTCGGA

Colony assay. FACS-sorted CD31⁺c-Kit⁺ cells from infected AGMs wereplated in MethoCult™ M03434 complete medium (StemCell Technologies) in a35 mm dish. After incubation in a humidified incubator at 37° C. with 5%carbon dioxide for 7 days, colonies were visualized by microscopy andquantitated. Cells isolated from colonies were subjected toWright-Giemsa staining.

Quantitative real-time RT-PCR. Total RNA was purified from TRIzol®(Invitrogen) according to manufacturers' instructions. cDNA wassynthesized from 1 μg purified total RNA by Moloney murine leukemiavirus reverse transcriptase (M-MLV RT). Real-time PCR was performed withSYBR green master mix (Applied Biosystems) and Product accumulation wasmonitored by SYBR green fluorescence using either a StepOnePlus™ orViia™7 instrument (Applied Biosystems). Relative expression wasdetermined from a standard curve of serial dilutions of cDNA samples,and values were normalized to 18S RNA expression. The sequences ofprimers used for RT-PCR and genotyping are provided below.

Flow cytometry. Dissociated cells from cultured AGM explants wereresuspended in PBS containing 2% FBS and passed through 25-μm cellstrainers to obtain single-cell suspensions prior to antibody staining.Fetal liver cells were washed with PBS once before antibody staining.APC-conjugated antibody CD31 (MEC13.3, Biolegend), PE-conjugatedantibody c-Kit (2B8, eBioscience), PerCP-Cy5.5-conjugated antibody Sca1(D7, eBioscience), APC-conjugated antibody Ter119 (TER119, eBioscience)and PE-conjugated CD71 (R17217, eBioscience) were used for flowcytometry and Fluorescence Activated Cell Sorting (FACS). Samples wereanalyzed on a FACSAria™ II cell sorter (BD Biosciences). Cells weregated on GFP to ensure retroviral expression. DAPI (Sigma-Aldrich)exclusion was utilized for live/dead discrimination. FACS-sorted cellswere immediately processed for RNA isolation or colony assay.

ChIP assay. Quantitative chromatin immunoprecipitation (ChIP) wasconducted as previously described using antibodies specific formonomethylated H4K20 (Millipore), GATA-1, and Scl/TAL1. Samples wereanalyzed by quantitative real-time PCR using either a StepOnePlus™ orViia™ 7 instrument (Applied Biosystems). The amount of product wasdetermined relative to a standard curve generated from a serial dilutionof input chromatin.

Statistical analysis. Student's t-tests were conducted using GraphPadSoftware or Microsoft Excel. For all figures, *: p<0.05; **: p<0.01;***: p<0.001.

Primers. +9.5 flanking forward: (SEQ ID NO: 4)5′-ATGTCCTTTCGGATCTCCTGCC-3′ +9.5 flanking reverse: (SEQ ID NO: 5)5′-GGTAAACAGAGCGCTACTCCTGTGTGTT-3′ 18S rRNA forward: (SEQ ID NO: 6)5′-CGCCGCTAGAGGTGAAATTCT-3′ 18S rRNA reverse: (SEQ ID NO: 7)5′-CGAACCTCCGACTTTCGTTCT-3′ Gata mRNA forward: (SEQ ID NO: 8)5′-GCAGAGAAGCAAGGCTCGC-3′ Gata mRNA reverse: (SEQ ID NO: 9)5′-CAGTTGACACACTCCCGGC-3′ Gpr65 mRNA forward: (SEQ ID NO: 10)5′-CAAGAGAAGCATCCCTCCAGAA-3′ Gpr65 mRNA reverse: (SEQ ID NO: 11)5′-TGTTTTTATTTTCACGCCGTTTG-3′ Gata2 primary transcript forward:(SEQ ID NO: 12) 5′-GACATCTGCAGCCGGTAGATAAG-3′Gata2 primary transcript reverse: (SEQ ID NO: 13)5′-CATTATTTGCAGAGTGGAGGGTATTAG-3′ MyoD promoter forward: (SEQ ID NO: 14)5′-GGGTAGAGGACAGCCGGTGT-3′ MyoD promoter reverse: (SEQ ID NO: 15)5′-GTACAATGACAAAGGTTCTGTGGGT-3′ Eifk promoter forward: (SEQ ID NO: 16)5′-GTGATTTCCTTCCAGCAGTTGTAA-3′ Eifk promoter reverse: (SEQ ID NO: 17)5′-CTCACGCTATTGGTCTCTTTTAAGTG-3′ Gata2_9.5 Site_933 bp forward:(SEQ ID NO: 18) 5′-CTTGCTGCTGGCTCTGAGAAC-3′Gata2_9.5 Site_933 bp reverse: (SEQ ID NO: 19)5′-AGTCCAGGGTCTTTTAAGGATAAATTC-3′ Gata2_9.5 Site_480 bp forward:(SEQ ID NO: 20) 5′-AACCTTCAAATGCAGACACTTCAC-3′Gata2_9.5 Site_480 bp reverse: (SEQ ID NO: 21)5′-GAATCCGCCAGAACGAAGAC-3′ Gata2_9.5 Site forward: (SEQ ID NO: 22)5′-GACATCTGCAGCCGGTAGATAAG-3′ Gata2_9.5 Site reverse: (SEQ ID NO: 23)5′-CATTATTTGCAGAGTGGAGGGTATTAG-3′ Gata2_9.5 Site_446 bp forward:(SEQ ID NO: 24) 5′-GCCGAGGGAGTTCAGTGCTA-3′Gata2_9.5 Site_446 bp reverse: (SEQ ID NO: 25)5′-AGCGCTACTCCTGTGTGTTCTTC-3′ Gata2_9.5 Site_880 bp forward:(SEQ ID NO: 26) 5′-TCCTGGCGACTCCTAGATCCTA-3′Gata2_9.5 Site_880 bp reverse: (SEQ ID NO: 27)5′-GAAAGCCCTGAGGAAGTTGGA-3′ Lyl1 Exon 1 forward: (SEQ ID NO: 28)5′-TCAGCATTGCTTCTTATCAGCC-3′ Lyl1 Exon 1 reverse: (SEQ ID NO: 29)5′-CGCAGAGGCCAGAGGATG-3′ Kit_114 kb forward: (SEQ ID NO: 30)5′-GCACACAGGACCTGACTCCA-3′ Kit_114 kb reverse: (SEQ ID NO: 31)5′-GTTCTGAGATGCGGTTGCTG-3′ Hdc mRNA forward: (SEQ ID NO: 32)5′-ACCTCCGACATGCCAACTCT-3′ Hdc mRNA reverse: (SEQ ID NO: 33)5′-CCGAATCACAAACCACAGCTT-3′ c-Kit mRNA forward: (SEQ ID NO: 34)5′-AGCAATGGCCTCACGAGTTCTA-3′ c-Kit mRNA reverse: (SEQ ID NO: 35)5′-CCAGGAAAAGTTTGGCAGGAT-3′ Lyl1 mRNA forward: (SEQ ID NO: 36)5′-AAGCGCAGACCAAGCCATAG-3′ Lyl1 mRNA reverse: (SEQ ID NO: 37)5′-AGCGCTCACGGCTGTTG-3′ c-Myb mRNA forward: (SEQ ID NO: 38)5′-CGAAGACCCTGAGAAGGAAA-3′ c-Myb mRNA reverse: (SEQ ID NO: 39)5′-GCTGCAAGTGTGGTTCTGTG-3′ Runx1 mRNA forward: (SEQ ID NO: 40)5′-TCACTGGCGCTGCAACAA-3′ Runx1 mRNA reverse: (SEQ ID NO: 41)5′-TCTGCCGAGTAGTTTTCATCGTT-3′ TAL1 mRNA forward: (SEQ ID NO: 42)5′-GAGGCCCTCCCCATATGAGA-3′ TAL1 mRAN reverse: (SEQ ID NO: 43)5′-GCGCCGCACTACTTTGGT-3′ Sfpi1 mRNA forward: (SEQ ID NO: 44)5′-GGCAGCGATGGAGAAAGC-3′ Sfpi1 mRNA reverse: (SEQ ID NO: 45)5′-GGACATGGTGTGCGGAGAA-3′

Example 1: GATA-2 Expression in +9.5^(−/−) AGM Rescues HematopoieticCell Generation

Deletion of the +9.5 site reduced Gata2 expression in AGM hemogenicendothelium and abrogated EHT (FIG. 1A). These results suggest thatfactors/signals conferring +9.5 activity control Gata2 expression, whichpromotes EHT. In principle, the +9.5 might regulate other genes in cisor in trans that control EHT. To determine if the HSC generation defectof +9.5 AGM results from insufficient GATA-2 production, we testedwhether GATA-2 expression rescues the defect. E11.5 mouse embryo AGMswere infected with GATA-2-expressing retrovirus (FIG. 1A). After 96 h ofexplant culture, we quantitated endothelial and hematopoietic cellpopulations in infected (GFP⁺) cells by flow cytometry using CD31 andc-KIT surface markers. The infection efficiency was similar among thethree conditions (+9.5^(+/+)-empty vector, +9.5^(−/−)-empty vector,+9.5^(−/−)-GATA-2), as indicated by the indistinguishable percentage ofGFP⁺ live cells (FIG. 1B). GATA-2 expression restored Gata2 mRNA to thewild type level in CD31⁺c-KIT⁻ endothelial and CD31⁺c-KIT⁺ hematopoieticcells (FIG. 1C), and rescued both populations (FIGS. 1D and 1E). Sincethe infected cells express GFP, the percentage of GFP⁺ cells in eachpopulation increased in +9.5^(−/−) AGM infected with GATA-2-expressingretrovirus (FIG. 1F). However, retroviral-mediated GATA-2 expression didnot rescue CD31⁺c-KIT⁻ endothelial and CD31⁺c-KIT⁺ hematopoietic cellpopulations in the GFP⁻ cells (data not shown). Thus, the +9.5^(−/−) AGMhematopoiesis defect resulted from insufficient GATA-2 production, isassociated with reduced CD31⁺c-KIT⁻ endothelial cells, and can berectified by restoring GATA-2.

To assess whether the GATA-2-rescued CD31⁺c-KIT⁺ hematopoietic cells arefunctional, their capacity to generate myelo-erythroid colonies in aColony Forming Unit (CFU) assay was determined. After culturingretroviral-infected AGMs for 96 h, GFP⁺ CD31⁺c-KIT⁺ hematopoietic cellswere isolated by fluorescence activated cell sorting (FACS) and assayedfor their capacity to generate BFU-E, CFU-GM and CFU-GEMM colonies.Whereas +9.5^(−/−) AGM failed to generate CFUs, GATA-2 expression in the+9.5^(−/−) AGM induced CFUs comparable to +9.5^(+/+) AGM (FIG. 2A).Quantification of colony types revealed comparable numbers of rescuedCFU-GM colonies in comparison with wild type AGM (FIG. 2B). GATA-2expression induced BFU-E and CFU-GEMM colonies (FIG. 2B). Coloniesderived from wild type and rescued samples were morphologicallyindistinguishable (FIG. 2C). Wright-Giemsa staining of cells fromcolonies revealed normal myeloid and erythroid cell generation from theGATA-2-expressing +9.5^(−/−) AGM (FIG. 2D). The GATA-2-inducedCD31⁺c-KIT⁺ hematopoietic cells exhibited qualitatively andquantitatively normal activity.

Example 2: Global GPCR Analysis in the AGM: Discovery of a GATAFactor-Regulated GPCR Cohort

The +9.5 site confers Gata2 expression and establishes a genetic networkinvolving known HSC regulators and genes not implicated inhematopoiesis. To discover vital constituents of the network, especiallythose with potential for modulation by small molecules/drugs, wesystematically analyzed the expression pattern of non-olfactory GPCRs(FIG. 3A). The human genome encodes greater than 800 GPCRs, which arecategorized into rhodopsin, secretin, glutamate, adhesion andfrizzed/taste2 families based on sequence homology; 341 are distinctfrom the olfactory and taste GPCRs. Using AGM RNA-seq data, we parsedAGM-expressed GPCRs into the canonical categories: secretin, 5% (15);adhesion, 7% (22); glutamate, 5% (15); frizzled/taste2, 6% (20); andrhodopsin, 70% (221) (FIG. 3B).

To discover GPCRs that control HSC generation and/or activity, weevaluated GPCR expression in the AGM. Of the 314 GPCRs annotated byRNA-seq, 85 were expressed at >5 transcripts per million (TPM) (FIG.3C), a level that can be validated with high frequency by real-timeRT-PCR. Of the 85 GPCRs, 20 were downregulated in the +9.5^(−/−) AGMversus +9.5^(+/+) AGM (FIG. 3D), indicating GATA-2-regulation. Using ourprevious microarray dataset (untreated or β-estradiol-treatedG1E-ER-GATA-1 erythroid precursor cells), we found that 4 of the 20GATA-2-regulated GPCRs were GATA-1-regulated (FIG. 3D). Our strategyrefined the 314 non-olfactory, AGM-expressed GPCRs to yield Adora3,Gpr65, Ltb4r1, and P2ry1, which are GATA-2- and GATA-1-regulated.

Example 3: A GATA Factor-GPCR Feedforward Loop Suppresses AGMHematopoiesis

As shared gene expression patterns can infer functionalinterconnectivity, we compared Adora3, Gpr65, Ltb4r1, P2ry1, and Gata2expression patterns in mouse cells and tissues. Only Gpr65 resembledGata2, both being expressed in HSPCs and mast cells (data not shown). AsGATA-1 represses Gata2 during erythroid maturation via a GATA switch,Gata2 expression declines upon erythroid maturation. Mining the ErythronDatabase, which provides transcriptomics data during erythroid precursorcell maturation into erythrocytes, revealed Gata2 and Gpr65 repressionupon erythroid differentiation. Gpr65 was the only one of the four GPCRgenes to have a Gata2-like expression pattern (FIG. 3E). To furthercompare expression patterns, we quantitated Gata2 and Gpr65 mRNA inG1E-ER-GATA-1 cells treated with β-estradiol to induce erythroidmaturation and in FACS-sorted R1, R2, R3 and R4/5 fetal liver cellpopulations. Gpr65 and Gata2 were repressed during erythroid maturation(FIGS. 3F and 3G). These correlations are consistent with GATA-2upregulating Gpr65 expression in the AGM and may point to a functionallink between GATA-2 and GPR65. ChIP-seq analysis in human (FIG. 3H) andmouse (FIG. 3I) cells revealed GATA-2 occupancy at Gpr65, suggestingthat GATA-2 directly regulates Gpr65 transcription. Comparison of+9.5^(+/+) and +9.5^(−/−) AGM revealed that reduced Gata2 expression inthe +9.5^(−/−) AGM decreased Gpr65 expression, and Gpr65 expression wasundetectable in the yolk sac (FIG. 3J). Previously, we demonstrated thatGATA-2 expression in Mouse Aortic Endothelial cells increasestranscription of certain GATA-2 target genes. In this system, GATA-2increased Gpr65 expression (FIG. 3K), indicating that GATA-2 regulatesGpr65 expression in multiple contexts. To analyze the Gpr65 expressionpattern in distinct cell types within the AGM, we mined RNA-seq dataobtained with FACS-sorted endothelial cells (EC), hemogenic endothelialcells (HEC), hematopoietic cells (HC) and hematopoietic stem cells (HSC)from the AGM (Solaimani Kartalaei et al., 2015). This analysis revealedthat Gpr65 is detectable in all cell types, and the levels are lower inHSCs (FIG. 3L).

To test whether GPR65 controls hematopoiesis in the AGM, we conducted aloss-of-function analysis using a Gpr65 shRNA retrovirus. E11.5 AGMswere infected, and after culturing for 96 h, hematopoietic cellpopulations were quantitated by flow cytometry. Quantitation of GFP⁺live cells indicated that control shRNA (shLuc) and shGpr65 retroviruseshad an indistinguishable infection efficiency (FIGS. 4A and 4B). Gpr65knockdown reduced Gpr65 mRNA by 60-70% (FIG. 4C). While downregulatingGpr65 did not alter CD31⁺c-KIT⁺ hematopoietic cells (FIGS. 4A and 4D),it increased CD31⁺c-KIT⁺SCA1⁺ HSC-containing, multipotent hematopoieticcells (FIGS. 4A and 4E). Retroviral-mediated Gpr65 shRNA expression didnot alter CD31⁺c-KIT⁺ hematopoietic cells and CD31⁺c-KIT⁺SCA1⁺HSC-containing cells in GFP⁻ cells (data not shown). These resultsindicate that GPR65 suppresses hematopoiesis in the AGM.

To assess whether Gpr65 activity to suppress hematopoiesis operates inother systems, we used a morpholino (MO) targeting the Gpr65 translationstart site (Gpr65_ATG MO) to reduce Gpr65 expression in zebrafishembryos. The Gpr65_ATG MO was injected into 1-cell stage embryos, whichwere analyzed for expression of the HSC markers Runx1/cmyb using in situhybridization (ISH) 36 h post-fertilization. The Gpr65_ATG MOdose-dependently increased expression of the HSC markers Runx1/cmyb inthe embryos (FIGS. 4F and 4G). A second MO, which blocks Gpr65 splicing(Gpr65_SP MO) yielded an identical result; Gpr65 downregulation inducedexpression of the HSC markers Runx1/cmyb (FIG. 4G). Thus, GPR65suppresses hematopoiesis in mouse and zebrafish embryos.

shRNA and morpholino-based loss-of-function strategies may be confoundedby off-target effects. As an alternative strategy we used a GPR65antagonist, the lysosphingolipid galactosylsphingosine (psychosine).Psychosine was initially proposed to be a GPR65 agonist, based on theGPR65 requirement for psychosine-induced multi-nuclear cell formation.Subsequently, it was demonstrated that GPR65 is a proton-sensingreceptor, and psychosine antagonizes GPR65. We treated E11.5 AGMs withvehicle or psychosine, and after 96 h, hematopoietic cells werequantitated by flow cytometry. While psychosine did not alterCD31⁺c-KIT⁺ cells (FIGS. 5A and 5B), it increased theCD31⁺c-KIT⁺SCA1⁺cell population, which is known to contain multipotenthematopoietic precursors (FIGS. 5A and 5C). In aggregate, the mouse andzebrafish studies indicate that GPR65 is an endogenous suppressor ofhematopoiesis.

Example 4: GPR65 Establishes Repressive Chromatin and Disrupts anActivating Complex on a Cis-Element Required for Gata2 Transcription

To elucidate the mechanism underlying GPR65 suppression ofhematopoiesis, we considered whether GPR65 might downregulate keyregulators of HSC generation/activity. After knocking down GPR65, weisolated infected CD31⁺c-KIT⁻ endothelial cells that give rise to HSCs.Downregulating Gpr65 mRNA by 60-70% increased Gata2 mRNA 2.9 fold(p=0.03) and its downstream target Runx1 mRNA (p=0.04) 2.9-fold (FIG.5D). The knockdown elevated Gata2 primary transcripts 3.9-fold (p=0.04)(FIG. 5E), indicating that GPR65 suppresses Gata2 transcription.

To determine whether GPR65 regulates Gata2 expression in zebrafish, weanalyzed Gata2 expression using ISH 36 h post-fertilization. Zebrafishhas two Gata2 homologs; Gata2b is enriched in hemogenic endothelium andregulates HSPC emergence. Whereas the majority of uninfected embryosexhibited broad staining in the AGM, there was no clear linear zoneenriched in hemogenic endothelium. However, Gpr65_ATG MO injectedembryos exhibited the linear zone (data not shown), suggesting thatGPR65 suppresses Gata2 expression in zebrafish embryos. Given thatGATA-2 promotes HSC emergence in the AGM and regulates HSC activity, wepropose that GPR65 suppresses hematopoiesis by limiting Gata2 expressionand GATA-2 levels.

As Gpr65 is expressed in AGM endothelium and HSPCs, we asked whetherGPR65 represses Gata2 expression in other contexts. Fetal liverhematopoietic precursors express Gata2, and as GATA-1 rises uponerythroid maturation, Gata2 is repressed. We isolated Lin⁻ hematopoieticprecursors from E14.5 fetal livers and tested whether reducing Gpr65expression with the Gpr65 shRNA retrovirus alters Gata2 expression.Cells were expanded for 72 h to increase HSPCs, while suppressingdifferentiation. Downregulating Gpr65 increased theproerythroblast-enriched R2 population 1.9-fold (p=0.0002) and reducedthe R3 population (early and late basophilic erythroblasts) 1.6-fold(p=0.0003) (FIG. 6A). Increased R2 cells, concomitant with reduced R3cells, suggests that GPR65 promotes erythroid maturation. DownregulatingGpr65 mRNA by 70-80%, which lowered GPR65 protein by 50%, increasedGata2 mRNA and primary transcripts 2.4 (p=3.65E-06) and 2.8 fold(p=0.014), respectively (FIGS. 6B and 6C). Western blot analysis offetal liver cells revealed that reducing GPR65 expression upregulatedGATA-2 (FIG. 6D). Increased GATA-2 selectively elevated GATA-2 targetgene expression (FIG. 6E). To test whether increased Gata2 expressionreflected a change in cellularity, we used FACS to isolate theGATA-2-expressing R2 population and compared gene expression in controland knockdown R2 cells. Gpr65 knockdown upregulated Gata2 mRNA 2.3 fold(p=0.02), primary transcript 4.8 fold (p=0.01), GATA-2 protein, andGATA-2 target genes in FACS-sorted R2 cells (FIGS. 6F, 6G, 6H and 6I).These results indicate that GPR65 represses Gata2 in the AGM and fetalliver. Since GATA-1 needs to repress Gata2 transcription early inerythroid maturation, upregulated Gata2 expression caused by the Gpr65knockdown would be expected to increase immature R2 cells as observed(FIG. 6A).

The +9.5 enhances Gata2 expression in the AGM and fetal liver. Deletingthe +9.5 abrogated HSC generation in the AGM and disrupted establishmentof the HSPC compartment in the fetal liver. To determine if increasedGata2 transcription upon Gpr65 knockdown requires the +9.5, we isolatedLin⁻ HSPCs from E14.5 +9.5^(+/−) fetal livers and infected cells withGpr65 shRNA retrovirus. After 72 h of expansion culture, allele-specificprimers were used to quantitate primary transcripts from wild type (WT)and mutant (Mut) 9.5 alleles in fetal liver cells and FACS-purified R2cells (FIG. 6J). Gpr65 knockdown upregulated Gata2 primary transcriptsfrom the wild type, but not the +9.5 mutant, allele (FIG. 6J),demonstrating importance of the +9.5 for Gata2 transcription.

Previously, we demonstrated that SetD8, the enzyme that monomethylatesH4K20, represses Gata2 expression via the +9.5 site. We tested therelevance of this mechanism to GPR65-mediated Gata2 repression. Weconducted quantitative chromatin immunoprecipitation (ChIP) analysis forH4K20me1 in fetal liver cells (control or Gpr65 knockdown) afterculturing for 72 h. Downregulating Gpr65 did not alter the SetD8 mRNAlevel (FIG. 6K). Gpr65 downregulation reduced H4K20me1 at the +9.5 site(p=0.002) and at sites 480 bp upstream (p=0.002), 466 bp downstream(p=0.006), and 880 bp downstream (p=0.02) of the +9.5 site (FIG. 6K).H4K20me1 was unaltered at the repressed muscle-specific MyoD promoterand the constitutively active Eif3k promoter. These results support amechanism in which GPR65 represses Gata2 by increasing a repressivechromatin mark at the +9.5 site. The basic helix-loop-helixtranscription factor Scl/TAL activates Gata2 transcription through the+9.5 site, and Scl/TAL1 chromatin occupancy is reduced at the +9.5 siteduring GATA-1-mediated Gata2 repression. As Gpr65 downregulationdecreased H4K20me1 at the +9.5 site, this alteration may generatechromatin that is more accessible to cognate binding factors. Todetermine if GPR65 alters GATA-1 and Scl/TAL1 occupancy, we quantitatedGATA-1 and Scl/TAL1 occupancy at the +9.5 site in fetal liver cellsinfected with control shRNA or Gpr65 shRNA-expressing retrovirus. Whileknocking down Gpr65 did not alter GATA-1 occupancy at the +9.5, theknockdown increased Scl/TAL1 occupancy at the +9.5 1.6-fold (p=0.02) butnot at other target loci (Lyl1 and Kit) (FIG. 6K and data not shown).These results suggest that GPR65 represses Gata2 by establishingrepressive chromatin that limits Scl/TAL1 occupancy at the +9.5 site(FIG. 6L).

Example 5: Psychosine Regulates Gata2 Expression

To test whether psychosine regulates Gata2 expression, we isolated Lin⁻hematopoietic precursors from E14.5 fetal livers and treated cells withpsychosine. After 72 h of ex vivo culture, flow cytometry was used toquantitate erythroid maturation. This analysis revealed that treatmentwith psychosine dissolved in DMSO slightly, but consistently, increasedR2 cells and decreased R3 cells in comparison to cells treated with DMSOalone (control) (FIG. 7A). suggesting that psychosine has some capacityto inhibit erythroid maturation. RNA analysis revealed that Gata2 mRNAand primary transcript levels were 2-3 fold higher in the fetal livercells treated with psychosine versus the DMSO-treated control cells(FIG. 7B), indicating that psychosine increases Gata2 expression infetal liver cells. To test whether increased Gata2 expression reflecteda change in cellularity or increased Gata2 transcription independent ofpotential cellular changes, fluorescence activated cell sorting (FACS)was used to isolate the GATA-2-expressing R2 population. We comparedGata2 expression in control and psychosine-treated, isolated R2 cells.RT-PCR and Western blot analysis revealed that psychosine increasedGata2 mRNA, Gata2 primary transcript (unprocessed mRNA that serves as ametric of transcription), and GATA-2 protein in R2 cells (FIGS. 7C and7D).

Example 6: Activity of Psychosine Derivatives

Furthermore, we analyzed the activity of structurally distinctderivatives of psychosine (glucosylsphingosine and Lactosylsphingosine),specifically, the role of the sugar constituent in the fetal liver (FIG.8). To determine the activity of psychosine derivatives, we treated theLin-HSPCs with DMSO as control, psychosine from Sigma or Avanti,glucosphingosine and lactosphingosine. Flow analysis revealed thatglucosylsphingosine, but not lactosylsphingosine exhibited similaractivity as psychosine, which slightly but significantly blockserythropoiesis (FIGS. 9A and 9B). RNA and protein analysis showed thatglucosylsphingosine has similar activity with psychosine, thatupregulated Gata2 expression at RNA and protein level (FIGS. 9C and 9D).

Example 7: Aorta, Gonad, Mesonephros (AGM) Culture and Transplant Assay

AGM explant culture. E11.5 AGMs are dissected and cultured by methodsknown in the art. In brief, intact AGMs are cultured for 4 d on Duraporefilters (Millipore) at the air-liquid interface in IMDM+ Media (Iscove'smodified Dulbecco's media [IMDM; Invitrogen] supplemented with 20% FBS[Gemini], 4 mM 1-glutamine [Invitrogen], 1% penicillin/streptomycin[Invitrogen], 0.1 mM mercaptoethanol, 100 ng/ml IL-3 [R&D Systems], 100ng/ml Flt3L [R&D Systems], and 1.5% conditioned medium from a kitligand-producing CHO cell line).

AGM reaggregate culture. AGMs are dissociated and reaggregated forculturing by methods known in the art. A single-cell suspension of AGMcells is drawn into a 200-μl pipette tip. The tip containing the cellsuspension is blocked with Parafilm and centrifuged in a 15-mlcentrifuge tube at 300 g for 5 min. After removing the Parafilm, thereaggregate is extruded onto the Durapore filter and cultured at theair-liquid interface in IMDM+ media for 4 d. AGM reaggregates, as wellas whole AGM explants, are cultured in a humidified incubator at 37° C.with κ% carbon dioxide. Uncultured AGMs and cultured AGMexplants/reaggregates are digested in 0.1% collagenase in PBS containing10% FBS at 37° C. for 30 min and then dissociated by passing through a27-G needle. Dissociated AGM cells were subjected to colony assay,transplantation assay, flow cytometry, and cell sorting.

Transplantation assay. Adult C57BL/6 recipient mice (CD45.1+; 6-8 wkold) are lethally irradiated with 2 doses of 500 rad each from a Cesiumsource. Dissociated cells from E11.5 cultured CD45.2+ AGM explants arecombined based on their genotypes. 50,000 nucleated AGM cells wereco-injected into individual irradiated CD45.1+ recipient mice with200,000 CD45.1+ spleen cells as a support and 20,000 CD45.1+ bone marrowcells. The transplanted recipient mice are maintained ontrimethoprim/sulfamethoxazole-treated water for 2 wk. Blood is obtainedfrom the retroorbital venous sinus regularly after transplantation forflow cytometric analysis, as known in the art. Directly conjugatedantibodies specific for the following surface antigens are purchasedfrom eBioscience: CD19 (eBio1D3), CD45.1 (A20), CD45.2 (104), Mac-1(M1/70), and Thy1.2 (53-2.1).

Discussion

Genetic networks orchestrating stem and progenitor cell transitions canbe deconstructed into regulatory modules termed network motifs. Whereasconsiderable progress has been made to identify individual geneconstituents of genetic networks controlling hematopoiesis, manyquestions remain unanswered regarding how the numerous genes involvedform network motifs, how the network motifs amalgamate into the intactcircuitry, and whether the circuitry has an inherent plasticity andundergoes remodeling in states of altered hematopoiesis such as stress,aging, and malignancy.

Since the GATA-2-dependent genetic network promotes hematopoiesis, itseems reasonable to infer that GATA-2-induced factors are positivemediators of key steps in this process, including EHT, HSPCself-renewal, and HSPC differentiation. In erythroid cells, GATA-1activates heme biosynthetic genes, globin subunits, and constituents ofthe red cell cytoskeleton, all required for erythroid maturation.Moreover, GATA-1 represses Gata2, Lyl1, and Kit, as a means of enablingerythroid maturation. GATA-2 activates Kit, an essential regulator ofHSPCs, consistent with GATA-2 promoting HSPC development and function.

Using a coupled bioinformatics-experimental strategy, we analyzed thelarge GPCR gene family, with the goal to discover GATA-2-induced GPCRsthat promote HSPC transitions. This analysis led to the surprisingfinding that GATA-2 activates Gpr65 expression, which in turn suppresseshematopoiesis via negative feedback on the Gata2 gene. This negativefeedback mechanism conforms to a type I incoherent feed-forward loop,which is defined as an input (GATA-2) that elicits an output (EHT orhematopoiesis) through positive (increased Gpr65 expression) andnegative (GPR65 suppression of hematopoiesis) paths. Type I incoherentfeed-forward loops shape the dynamics of the respective mechanism, withthe existing paradigm assuming that the feed-forward loop confers apulse of activity to accelerate the reaction. Since the regulatoryconstituents and associated network motifs governing HSPC transitionsare still being identified, the dynamics of specific steps in thesetransitions are largely unexplored. The mechanism of GPR65-mediatedGata2 repression involved elevation of a repressive histone mark,H4K20me1, associated with reduced occupancy by the activator Scl/TAL1.In principle, GPR65 might directly target chromatin to restrict Scl/TAL1occupancy or might reduce Scl/TAL1 occupancy prior to chromatinmodification. Regardless of the order-of-events in this mechanism, theanalysis provided herein established a GATA factor-GPCR-dependent type Iincoherent feed-forward loop that suppresses, rather than promoteshematopoiesis.

Gpr65, originally identified as T-cell Death-Associated Gene-8 (Tdag8),is a proton/acid-activated GPCR. Whereas GPR65 had not been linked tothe control of HSPC transitions, it was reported to be a pro-apoptoticfactor in glucocorticoid-induced lymphocyte apoptosis and to regulatecytokine production from macrophages. However, targeted deletion ofGpr65 revealed it was dispensable for glucocorticoid-induced thymocyteapoptosis in vivo and in vitro. These results were suggested to reflectredundancy with related pH-sensing GPCRs, including G2A, OGR1, and GPR4,which would create a formidable obstacle to dissect Gpr65 function invivo. Although the impact of the pH-sensing mechanism to GATA-2 functionin the AGM and fetal liver is unclear, the endogenous GPR65 antagonistpsychosine resembled Gpr65 shRNA in upregulating Gata2 expression andhematopoiesis.

As GPR65 suppressed HSPC generation from primary mouse AGM explants, itis instructive to consider potential consequences of ablating thismechanism in vivo. Abrogation of the mechanism would upregulate HSPCgeneration, which might expand the hematopoietic compartment. However,given the capacity of HSPCs to compensate for hematopoieticperturbations in certain contexts, HSPC upregulation might be normalizedto ensure physiological homeostasis. Nevertheless, since ablating Gpr65would be expected to increase, rather than decrease HSPCs, evaluatingthe role of the GATA-2-GPR65 circuit in vivo will require a carefulquantitative analysis of HSPCs. Theoretical considerations of type Iincoherent feed-forward loops would suggest an alteration of thedynamics of specific steps of hematopoiesis, which might not be evidentfrom steady-state analyses. It is attractive to propose that thissuppressive mechanism is particularly important when the demand forhematopoiesis increases, e.g. during stress, since an unopposed increasecould yield deleterious blood cell elevations.

In summary, the present inventors have discovered that GATA-2 activityto promote hematopoiesis involves induction of both positive andnegative mediators, the balance of which establishes the physiologicaloutput. Based on the GPR65 pH-sensing function, one can developtechnologies to analyze the pH microenvironment of the AGM and otheranatomical sites of hematopoiesis and to establish the role of GPR65 asan endogenous mediator of pH-dependent, dynamic alterations in HSPCtransitions.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of expanding a population ofhematopoietic stem cells, comprising contacting the population ofhematopoietic stem cells with an effective amount of an inhibitor ofG-protein coupled receptor 65 (GPR65), and culturing the cells toprovide a population of expanded, substantially undifferentiatedhematopoietic stem cells, wherein the inhibitor of G-protein coupledreceptor 65 (GPR65) is a glucosphingolipid or a galactosphingolipid, ora salt of one of the foregoing inhibitors.
 2. The method of claim 1,wherein the method is performed ex vivo.
 3. The method of claim 1,wherein the population of hematopoietic stem cells is obtained from amammalian tissue selected from umbilical cord blood, peripheral blood,or bone marrow.
 4. The method of claim 1, wherein the population ofhematopoietic stem cells is obtained from mammalian umbilical cordblood.
 5. The method of claim 1, wherein the population of hematopoieticstem cells are of human origin.
 6. The method of claim 1, wherein theinhibitor of G-protein coupled receptor 65 (GPR65) is psychosine orglucosylsphingosine.
 7. A method for administering an expandedpopulation of hematopoietic stem cells to a patient in need thereof,comprising culturing a population of hematopoietic stem cells in ahematopoietic stem cell expansion medium ex vivo for a period of timesufficient to provide an expanded population of hematopoietic stemcells, wherein the hematopoietic stem cell expansion medium comprises aninhibitor of G-protein coupled receptor 65 (GPR65), and administeringthe expanded population of hematopoietic stem cells to the patient,wherein the inhibitor of G-protein coupled receptor 65 (GPR65) is aglucosphingolipid or a galactosphingolipid, or a salt of one of theforegoing inhibitors.
 8. The method of claim 7, wherein the populationof hematopoietic stem cells is obtained from a mammalian tissue selectedfrom umbilical cord blood, peripheral blood, or bone marrow.
 9. Themethod of claim 7, wherein the population of hematopoietic stem cells isobtained from mammalian umbilical cord blood.
 10. The method of claim 7,wherein the hematopoietic stem cells are human hematopoietic stem cellsand the patient is a human patient.
 11. The method of claim 7, whereinthe inhibitor of G-protein coupled receptor 65 (GPR65) is psychosine orglucosylsphingosine.
 12. The method of claim 7, wherein the patient isin need of treatment for leukemia, multiple myeloma, lymphoma, graftversus host disease, aplastic anemia, sickle cell anemia, SevereCombined Immune Deficiency (SCID), Wiskott-Aldrich Syndrome (WAS), IPEXSyndrome, Hemophagocytic Lymphohistiocytosis (HLH), X-linkedLymphoproliferative Disease (XLP) or Chronic Granulomatous Disease(CGD).